Bacillus anthracis is a Gram-positive, endospore-forming pathogen, which causes lethal inhalation anthrax in humans. It has also gained recent notoriety as a potential biothreat weapon. A hallmark of B. anthracis is the presence of two large plasmids pXO1 and pXO2 that carry the key virulence genes. Although pXO1 and pXO2 play major roles in the pathogenesis of anthrax, mechanisms of replication, segregation, and copy number control of these plasmids remain poorly characterized. Plasmid pXO1 contains a large pathogenicity island that houses genes encoding the tripartite anthrax toxin and several intregrases and transposases. The presence of mobile elements in pXO1 allows gene transfer to related Bacillus cereus family members. Therefore, the possibility of natural or engineered transfer of pXO1 to drug-resisant bacterial hosts presents a significant public health threat. The gap in our understanding of the mechanism of pXO1 maintenance in B. anthracis relates to the insignificant homology of pXO1-encoded proteins to known replication initiation or partitioning proteins elaborated by other plasmids. Recently, the pXO1 replicon was cloned and the plasmid-encoded RepX protein was found to be essential for mini-pXO1 replication in B. anthracis. Intriguingly, RepX shows significant homology to the tubulin-like bacterial FtsZ protein, which is widely conserved in the Prokaryotic Kingdom, and plays an essential cytoskeletal role in bacterial cell division. On this basis, it is hypothesized that RepX assembles into a GTP-regulated cytoskeletal structure essential for pXO1 replication/segregation and can be exploited as a novel target for pXO1 elimination. The goal of this proposal is to analyze if RepX assembles into GTP-dependent protein filaments in vitro and in vivo, and the role of RepX assembly in pXO1 replication/segregation. Another goal is to devise a high-throughput, forward chemical genetic screen to identify small molecules that will promote pXO1 loss by antagonizing RepX. The RepX inhibitors will be used as probes to study RepX and pXO1 dynamics in vivo and may constitute potential therapeutic leads. A subset of inhibitors may target a common site in RepX and FtsZ, and could thus serve as double-edged swords against anthrax.